INTRODUCTION

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Nitrosomonas europaea, a chemolithoautotrophic NH₃ oxidizer, oxidizes a variety of compounds, including alkanes, alkenes, alkynes (6, 10), halogenated hydrocarbons (12, 18, 27), and aromatic compounds (9, 13), with ammonia monooxygenase (AMO). AMO is the broad-substrate-range oxygenase that is responsible for oxidation of NH₃ to hydroxylamine (NH₂OH), the first step in oxidation of NH₃ to NO₂ (30). Previously described studies of cooxidation of halogenated hydrocarbons by NH₃-oxidizing bacteria have focused primarily on determining the range of compounds cooxidized by N. europaea (6, 7, 13, 16-18) and, to a lesser degree, on kinetic parameters (12). The majority of these studies were conducted by using short incubation periods (1 h), high-density cell suspensions (10⁹ to 10¹¹ cells ml¹) exhibiting high rates of NO₂ production (~3 µmol ml¹ h¹), and pH values considered to be optimal for NH₃ oxidation (pH 7.8 to 8.0). Comprehensive studies have not been performed yet with lower-density cell suspensions (<10⁸ cells ml¹) that exhibit NH₃ oxidation rates more typical of environments like nitrifying bioreactors (~0.1 µmol of NH₄⁺ ml¹ h¹) (1, 2), in which cooxidation may occur (15, 23). Furthermore, the sustainability of cooxidation and the relationship of cooxidation to NO₂ production could not be examined adequately in our previous studies because total ammonium (NH₄⁺ plus NH₃) became limiting very quickly because of high rates of consumption and because a decrease in pH reduced NH₃ availability. The issue of sustainability and the factors that affect it need to be studied in order to understand the long-term effects of cooxidation of halogenated hydrocarbons on NH₃ oxidizers and to better assess the potential use of these organisms in bioremediation of contaminants.

Methyl bromide (MeBr) is a soil fumigant that is used to control weeds, soilborne plant pathogens, and nematodes (25, 29, 31). MeBr has been categorized as a class 1 ozone-depleting chemical by the U.S. Environmental Protection Agency and is scheduled for complete phase-out within a few years (25). Thus, the fate of MeBr has some applied significance, and this compound also is an excellent model compound for examining cooxidation by N. europaea because the end products of MeBr cooxidation (formaldehyde and HBr) have been identified (11, 12, 17). The objective of this study was to examine cooxidation of MeBr by a moderately low-density suspension of N. europaea cells (~6 × 10⁷ cells ml¹) that oxidized NH₃ at a rate similar to the rates measured in nitrifying bioreactors (1, 2).

	MATERIALS AND METHODS

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Cell growth and preparation. Batch cultures (750 or 1,500 ml) of N. europaea ATCC 19718 were grown in Erlenmeyer or Fernbach flasks in the dark at 27°C with orbital shaking (150 rpm). The growth medium consisted of 25 mM (NH₄)₂SO₄ and other constituents as described elsewhere (4). Cells were harvested by centrifugation (11,000 × g, 15 min) after the late exponential phase was reached (3 days), washed twice in buffer (50 mM potassium phosphate, pH 7.2), and resuspended in buffer to an optical density at 660 nm of approximately 1.0. All assays were initiated with aliquots of this cell suspension within 1 h of preparation. Epifluorescence microscopic counting of 4',6'-diamidino-2-phenylindole (DAPI)-stained cells confirmed that the cell density was 1.2 × 10⁹ ± 0.3 × 10⁹ cells ml¹ when the optical density at 660 nm was 1.0. The average dry weight of the cell suspension was 0.32 ± 0.08 mg ml¹.

Preparation of assay vials. Portions (5 ml) of sterile assay buffer were added to sterile glass vials (capacity, 74 ml), which were sealed with gray butyl stoppers (Kimble, Owens, Ill.) and aluminum crimp top seals (Wheaton, Millville, N.J.). The assay buffer contained 5 mM (NH₄)₂SO₄ and 50 mM KH₂PO₄-K₂HPO₄ (pH 7.2) unless otherwise noted. An MeBr stock vial was prepared by flushing a sealed vial containing 5 to 10 glass beads for 1 min (with periodic shaking to disrupt air pockets) with MeBr (99.5% pure; Matheson Gas Products, Inc., Newark, Calif.) from a lecture bottle. After an overpressure of gas was established in the stock vial, appropriate amounts of MeBr were added to assay vials by using gas-tight Hamilton microsyringes equipped with sidebore needles. Experiments were initiated by adding an aliquot of the cell suspension to each vial so that the final cell density was 6 × 10⁷ ± 1.5 × 10⁷ cells ml¹ (16 ± 4 µg [dry weight] of cells ml¹, 80 ± 20 µg vial¹) unless otherwise noted. The vials were inverted and incubated in the dark at 27°C.

Analytical procedures. MeBr oxidation was measured by monitoring the disappearance of MeBr from the gas phase in the assay vials by using a Shimadzu model GC-14 gas chromatograph. The gas chromatograph was equipped with a stainless steel column (outside diameter, 0.32 cm; length, 91 cm) packed with Porapak-Q (80-100 mesh; Waters Associates Inc., Framingham, Mass.) (column temperature, 120°C) and a flame ionization detector (detector and injector temperature, 200°C) interfaced with a Shimadzu model CR501 integrator. At time intervals, 60- to 200-µl aliquots of headspace gas were removed from the assay vials with a gas-tight Hamilton microsyringe equipped with a sidebore needle. To check for AMO-independent oxidation of MeBr, we included control vials that contained 1% (vol/vol) acetylene, a specific mechanism-based inactivator of AMO (4). By using these controls we determined that about 10 and 20% of the MeBr that disappeared from vials in 24-h assays and 48- to 72-h assays, respectively, could be considered AMO independent. These quantities were routinely subtracted from the MeBr depletion values that were obtained with vials that did not contain acetylene. The amounts of MeBr in the vials were determined by comparison with standards containing known amounts of MeBr in 74-ml vials containing 5 ml of sterile assay buffer. A dimensionless Henry's Law constant for MeBr of 0.25 was utilized (24), and approximately 22% of the total amount of MeBr added to the vials partitioned into the liquid phase. For all assays the amount of MeBr added was expressed as the concentration in the liquid phase, while the amount transformed was expressed in micromoles. For purposes of comparison, when 10 µmol of MeBr was added to a vial, the aqueous phase concentration of MeBr was 0.44 mM. NO₂ production was determined by removing aliquots (20 to 100 µl) of cell suspensions from the sealed vials with a gas-tight syringe and determining the NO₂ contents colorimetrically (3).

Response of the initial rate of MeBr oxidation to cell density, pH, and NH₄⁺ and MeBr concentrations. (i) Cell density. Aliquots of diluted cell suspensions of N. europaea were added to triplicate assay vials containing buffer, 10 mM NH₄⁺, and 0.5 to 3 µmol of MeBr vial¹ (aqueous concentration, 0.02 to 0.13 mM) in order to obtain cell densities of 6 × 10⁶, 6 × 10⁷, and 6 × 10⁸ cells ml¹. Samples of headspace gas were recovered at 3- to 12-h intervals for up to 3 days and examined to determine if MeBr depletion could be measured.

(ii) pH. N. europaea cell suspensions were prepared in phosphate buffer preadjusted to pH 6.2, 7.2, or 8.2. One-milliliter aliquots (6 × 10⁷ cells ml¹) were injected into vials that already contained either 0.11, 0.22, or 0.44 mM MeBr and 10 mM NH₄⁺ in the appropriate buffer. Samples of the headspace gas and liquid contents of the vials were obtained and used to determine the MeBr and NO₂ contents at 3- to 6-h intervals over a 24-h period.

(iii) NH₄⁺ and MeBr concentrations. N. europaea cells (6 × 10⁷ cells ml¹) were incubated in a factorialized design experiment with combinations consisting of 0.11, 0.22, or 0.44 mM MeBr and 2.5, 5, or 10 mM NH₄⁺. MeBr oxidation and NO₂ production were monitored at 2- to 6-h intervals. The rates of MeBr oxidation and NO₂ production were calculated by using the linear regression feature in SigmaPlot 3.0 (Jandel Scientific, San Rafael, Calif.). The correlation coefficients for all regressions were 0.96. The initial rates were expressed in micromoles of MeBr oxidized or NO₂ produced per milligram (dry weight) per hour.

Examination of the factors that might influence the sustainability of MeBr oxidation. (i) Influence of buffer, growth medium, and Na₂CO₃ on the sustainability of MeBr oxidation. N. europaea cells (6 × 10⁷ cells ml¹) were incubated in either phosphate buffer or complete growth medium (pH 7.2) that was supplemented or not supplemented with Na₂CO₃ (4 mM). The pH of each solution was adjusted to 7.2, and 4 mM Na₂SO₄ was added to vials that did not receive Na₂CO₃. Both the cell suspensions and the NH₄⁺ stock solution were prepared by using the appropriate buffer or growth medium. MeBr disappearance was monitored over a 36-h period.

(ii) Influence of NH₄⁺ limitation or cell inactivation on the sustainability of MeBr oxidation. To determine if either NH₄⁺ limitation or cell inactivation was the primary reason for the loss of MeBr-oxidizing activity, a preparation containing 6 × 10⁷ cells ml¹ was incubated with 10 mM NH₄⁺ and 0.22 mM MeBr until the initial rate of MeBr oxidation declined (after approximately 9 h of incubation). Then replicate vials received either (i) additional cells (3 × 10⁸ cells), (ii) additional cells plus NH₄⁺ (equivalent to an additional 10 mM), or (iii) NH₄⁺ alone. MeBr oxidation and NO₂ production were monitored for another 15 h.

(iii) Effects of the end products of NH₃ and MeBr oxidation, NH₄⁺ depletion, and pH decline on sustainability of MeBr oxidation. The effects of accumulation of the end products of MeBr and NH₃ oxidation on the sustainability of MeBr oxidation were examined by monitoring the oxidation of 0.22 mM MeBr in 50 mM phosphate buffer (pH 7.0) supplemented with 7.5 mM NH₄⁺. Factorialized combinations consisting of 2.5 mM NO₂ and 0.4 mM formaldehyde were added to the assay vials; these concentrations were chosen because they represented the approximate conditions in the assay vials after 24 h of oxidation of 0.22 mM MeBr and 10 mM NH₄⁺. The reactions were started by adding 1-ml aliquots of cells suspended in phosphate buffer (pH 7.0), and MeBr disappearance and NO₂ production were monitored as described above.

(iv) Influence of inhibition of protein synthesis on NO₂ production during MeBr cooxidation. Either chloramphenicol (final concentration, 200 or 400 µg/ml) or kanamycin (10 to 50 µg/ml) was dissolved in 50 mM phosphate buffer (pH, 7.2). Aliquots of the buffer were then supplemented with 10 mM NH₄⁺ and injected into sealed vials containing enough MeBr so that the MeBr concentration in the aqueous phase was 0.22 or 0.44 mM. The reactions were started by adding N. europaea (6 × 10⁷ cells ml¹), and NO₂ production was monitored during 7 h of incubation.

Residual NH₄⁺- and NH₂OH-dependent O₂ uptake activity after oxidation of MeBr. To further examine the effect of MeBr oxidation on the residual activity of N. europaea, cells (6 × 10⁷ cells ml¹) were incubated with 10 mM NH₄⁺ and 0.11, 0.22, or 0.44 mM MeBr for 24 h. Following incubation, the vials were opened and vented for 5 min. The cell suspensions were filtered through 25-mm diameter 0.4-µm-pore-size polycarbonate filters and washed by filtering 9 ml of sterile buffer over the cells. Control vials containing cells, 10 mM NH₄⁺, and no MeBr were treated exactly like the vials that contained MeBr were treated. To determine residual O₂ uptake activity, the filters were placed in 2-ml portions of buffer, and the cells were washed off with gentle shaking. An aliquot of the cell suspension (1.6 ml, 64 µg [dry weight] of cells) was added to an O₂ electrode chamber. After 3 to 5 min of stirring, NH₄⁺ (final concentration, 10 mM) was added to the chamber, and the NH₄⁺-dependent O₂ uptake rate was measured over a 2- to 5-min interval. NH₄⁺-dependent O₂ uptake was stopped by adding 1-allyl-2-thiourea (final concentration, 0.1 mM), a reversible inhibitor of AMO (14). Subsequently, NH₂OH (final concentration, 0.6 mM) was added to the chamber to measure NH₂OH-dependent O₂ uptake.

Recovery of NO₂-producing ability by N. europaea after oxidation of 0.44 mM MeBr for 24 h. N. europaea (6 × 10⁷ cells ml¹) was exposed to 0.44 mM MeBr in phosphate buffer (pH 7.2) for 24 h as described above. Vials containing cells that were not exposed to MeBr were included as controls. Cells were harvested from the buffer, washed, and resuspended in complete growth medium at either pH 7.2 or pH 8. Unexposed cells were diluted another 10-fold in growth medium so that the initial rates of NO₂ production were similar for both exposed and unexposed cells. At 6-h intervals, samples of cells were recovered, and NO₂ production was determined as described above.

	RESULTS

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In preliminary experiments (data not shown) we established that oxidation of MeBr at concentrations up to 0.44 mM could be measured accurately with a cell density of 6 × 10⁷ cells ml¹ (16 µg [dry weight] of cells ml¹). Oxidation of MeBr at concentrations of >0.66 and 0.88 mM could be measured, but the rates decreased rapidly after only 1 to 4 h of incubation and thus were not studied in detail. At cell densities of <10⁷ cells ml¹, the incubation time required to accurately measure MeBr disappearance with a gas chromatograph equipped with a flame ionization detector was 24 h or more, and such determinations could be made only at low MeBr concentrations (0.04 mM). The MeBr-oxidizing ability of N. europaea was examined at three pH values (pH 6.2, 7.2, and 8.2) representing the range of pH values likely to be encountered in many natural environments. At each of the three MeBr concentrations evaluated (0.11, 0.22, and 0.44 mM) MeBr was oxidized significantly faster at pH 7.2 than at either pH 6.2 or pH 8.2 (data not shown). After 24 h of incubation the pH had changed very little in the pH 7.2 preparation (final pH, 7.0 to 7.1), which implied that the buffering capacity of 50 mM phosphate was adequate for dealing with the acidity generated during NH₃ oxidation by the concentrations of cells used for at least 24 h. Additional studies of the properties of MeBr oxidation by N. europaea were performed by using pH 7.2 and a cell density of 6 × 10⁷ cells ml¹.

By experimenting with moderately low cell densities we were able to examine the initial rates of MeBr oxidation and the accompanying rates of NO₂ production at different NH₄⁺ concentrations (Table 1). The highest rates of MeBr oxidation occurred in the presence of 2.5 to 10 mM NH₄⁺ and 0.22 to 0.44 mM MeBr. The responses of the initial rate of MeBr oxidation to NH₄⁺ concentration were different at different MeBr concentrations. In the presence of 0.11 mM MeBr, the rate of MeBr oxidation increased twofold, while the level of NO₂ production decreased threefold as the NH₄⁺ concentration decreased from 10 to 1 mM. In the presence of 0.22 mM MeBr, the initial rates of MeBr oxidation were relatively insensitive to changes in the NH₄⁺ concentration at concentrations between 1 and 10 mM, despite the fact that the level of NO₂ production changed fivefold over this concentration range. In the presence of 0.44 mM MeBr, the initial rate of MeBr oxidation responded to most incremental changes in the NH₄⁺ concentration and decreased at NH₄⁺ concentrations between 5 and 2.5 mM and between 2.5 and 1 mM. When we examined the ratio of amount of MeBr oxidized to amount of NO₂ produced (M/N ratio), we found that the maximum initial rates of MeBr oxidation occurred at almost identical M/N ratios (the M/N ratios were 0.24, 0.21, and 0.21 for MeBr concentrations of 0.44, 0.22, and 0.11 mM, respectively) regardless of the MeBr and NH₄⁺ concentrations. We also observed another trend: M/N ratios of 0.30 and <0.1 were associated with suboptimal initial rates of MeBr oxidation.

View this table: [in this window] [in a new window]   TABLE 1.   NO2 production and MeBr oxidation by N. europaea in the presence of different combinations of NH4+ and MeBra

The rates of MeBr oxidation invariably declined when the cells were incubated for more than 12 h, and they declined to zero within 12 to 24, 36 to 48, and 48 to 72 h in the presence of 0.44, 0.22, and 0.11 mM MeBr, respectively (Fig. 1a). Although the rate of NO₂ production was constant for at least 12 h in the control lacking MeBr, in the presence of 0.11, 0.22, and 0.44 mM MeBr the rates of NO₂ production were constant for approximately 12, 6, and 3 h, respectively, and then gradually declined to zero over ranges of time similar to the ranges of time as described above for MeBr oxidation (Fig. 1b). Whereas the rates of oxidation of 0.11, 0.22, and 0.44 mM MeBr definitely decreased at different times (36 to 48, 24 to 36, and 12 to 24 h, respectively), the corresponding rates of NO₂ production were very similar (2 to 3 µmol of NO₂ produced mg [dry weight] of cells¹ h¹). Despite relatively large differences in the rate and duration of active MeBr oxidation for the different NH₄⁺-MeBr combinations, similar amounts of MeBr were oxidized with a large number of the NH₄⁺-MeBr combinations (1 to 2 µmol per vial, 10 to 20 µmol mg [dry weight] of cells¹) (Table 2). In general, this was attributed to the fact that while the rates of MeBr oxidation were about two- to threefold lower when 0.11 MeBr was used than when 0.22 and 0.44 mM MeBr were used, the length of the period of active oxidation was inversely proportional to the MeBr concentration (e.g., 72, 48, and 24 h) (Table 2).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Time courses of MeBr consumption (a) and NO2 production (b) by N. europaea over a 48- to 72-h period. All vials contained 10 mM NH4+, phosphate buffer (pH 7.2), 6 × 107 cells of N. europaea ml1, and either no MeBr (), 0.11 mM MeBr (), 0.22 mM MeBr (), or 0.44 mM MeBr (). The error bars indicate the standard deviations of the means based on the results obtained for three replicate vials per treatment.

We obtained no evidence which supported the possibility that either (i) NO₂ and/or formaldehyde accumulation, (ii) a decrease in pH (pH 7.2 to 7.0), (iii) NH₄⁺ limitation (10 to 7.5 mM), or (iv) inadequate medium composition (buffer versus growth medium) was directly responsible for the decreases in NO₂-producing ability and MeBr-oxidizing ability. For example, when assays were initiated at pH 7 in the presence of 7.5 mM NH₄⁺ and different combinations of NO₂ (2.5 mM) and formaldehyde (0.3 mM), the characteristics and amounts of MeBr oxidized were similar to the characteristics and amounts obtained under typical assay conditions (Fig. 2). Furthermore, the initial rates of MeBr oxidation and NO₂ production could be sustained in the same assay vials for several additional hours if a second aliquot of cells (30 × 10⁷ cells) was added to the assay mixture after 9 h of incubation (Fig. 3). Although 10 mM NH₄⁺ added along with the cells increased the rate of NO₂ production, it did not increase the rate of MeBr oxidation to a value greater than the value obtained when only cells were added. The increase in NO₂ production without a concomitant increase in MeBr oxidation is consistent with other data which showed that the same rate of oxidation of 0.22 mM MeBr could be supported by a range of NH₄⁺ concentrations (2.5 to 20 mM NH₄⁺) (Table 1) (12) over which the rate of NO₂ production doubled.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Effects of NO2, formaldehyde, pH, and NH4+ on the MeBr consumed by N. europaea. All assay mixtures contained 6 × 107 cells of N. europaea ml1, and the initial conditions were as follows: pH 7.0, 7.5 mM NH4+, and 0.22 mM MeBr. NO2 and formaldehyde were added when appropriate to final concentrations of 2.5 and 0.5 mM, respectively. Symbols: , NO2 and formaldehyde both present; , NO2 present and formaldehyde absent; , NO2 absent and formaldehyde present; , NO2 and formaldehyde both absent. The error bars indicate the standard deviations of the means based on the results obtained for three replicate vials per treatment.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Effects of NH4+ and fresh cell supplements MeBr consumption (a) and NO2 production (b) by N. europaea. At time zero, all vials (except the control) contained 0.22 mM MeBr, 10 mM NH4+, and 3 × 108 cells of N. europaea. After 9 h of incubation, triplicate vials received 3 × 108 cells (), 3 × 108 cells plus 10 mM NH4+ (), or no additional supplement (). The control vials () were incubated without MeBr and did not receive an additional supplement. The error bars indicate the standard deviations of the means based on the results obtained for three replicate vials per treatment.

When cells were recovered from the incubation vials after 24 h of exposure to MeBr, approximately 80 to 90% of their NO₂-producing (data not shown) and NH₄⁺-dependent O₂ uptake (Table 3) activities had been lost. Much less of the whole-cell hydroxylamine (NH₂OH)-dependent O₂ uptake activity was lost (20 to 30%) after exposure to MeBr. Recovery of NO₂ production by MeBr-exposed cells was monitored after the cells were resuspended in fresh growth medium (pH 7.2 or 8) containing 20 mM NH₄⁺ (Fig. 4). Cells that were not exposed to MeBr but otherwise treated identically were diluted 10-fold to obtain a similar initial rate of NO₂ production, and these cells were used as a control. We found that the NO₂ concentration increased immediately in a nonlinear manner in both exposed and unexposed cells at both pH values. Although the amount of NO₂ produced during the first 6 h of incubation by the cells exposed to MeBr was about the same as the amount produced by the unexposed cells, the rate of NO₂ accumulation was lower in the former preparation. A 48- to 60-h recovery period was required before the cells exposed to MeBr exhibited the same rate of NO₂ production that they exhibited before they oxidized MeBr.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Development of NO2 production by N. europaea cells resuspended in fresh growth medium after they were exposed (solid symbols) or not exposed (open symbols) to 0.44 mM MeBr in phosphate buffer (pH 7.2). The growth medium contained 20 mM NH4+. Symbols: , growth medium, pH 7.2; , growth medium, pH 8; , growth medium, pH 7.2; , growth medium, pH 8. For clarity error bars are not shown. The standard deviations were 10% of the mean values regardless of the treatment.

	DISCUSSION

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By experimenting with moderately low-density cell suspensions we gained insight into characteristics of MeBr oxidation by N. europaea that were not detected in previous studies performed in our laboratory. For example, Rasche et al. (17) and Keener and Arp (12) used 0.5 to 4 mg (dry weight) of cells ml¹ in their analyses of MeBr oxidation by N. europaea. Because the capacity of N. europaea to transform MeBr is between 10 to 20 µmol of MeBr mg (dry weight) of cells¹, we know that the quantities of cells used by our colleagues could transform approximately 10-fold more MeBr than the amounts used routinely in these types of studies (2 to 10 µmol per assay mixture). It is not surprising, therefore, that they did not determine the finite capacity of N. europaea to oxidize MeBr and that NO₂ production and NH₄⁺-dependent O₂ uptake activities declined considerably as a consequence of prolonged MeBr oxidation.

At first we were confused by our finding that both NO₂-producing and MeBr-oxidizing activities were lost during transformation of MeBr because Rasche et al. (16) had concluded from short-term studies that monohalogenated aliphatic compounds could be degraded by N. europaea without AMO inactivation by the end products of cooxidation. Nonetheless, it has been reported that formaldehyde (28) and NO₂ in the absence of NH₄⁺ (20) inhibit NH₃ oxidation in N. europaea, yet we obtained no evidence that these end products were inhibitory to MeBr oxidation at the concentrations generated in our assays and under our experimental conditions. At this time, however, we cannot rule out the possibility that formaldehyde generated intracellularly might be more toxic to N. europaea than externally applied material is or that some oxidatively generated brominated chemical species might be the cause of toxicity. Although NH₄⁺-dependent O₂ uptake was reduced more severely by exposure to MeBr than NH₂OH-dependent O₂ uptake was reduced, our data indicate that prolonged MeBr oxidation resulted in a more general toxic effect on the cells than inactivation of AMO per se. For example, previous studies in our laboratory showed that when approximately 90% of the NH₄⁺-dependent O₂ uptake activity in N. europaea was eliminated by specifically inactivating AMO with strong light, NO₂-producing activity could be restored completely within 4 h of the time when cells were resuspended in fresh growth medium (8). In contrast, our studies showed that cultures exposed to MeBr for 24 h, in which approximately 90% of the NH₄⁺-dependent O₂ uptake activity was debilitated, required about 48 to 60 h of incubation to exhibit a rate of NO₂ production comparable to the initial rate detected in unexposed cells. Indeed, the effect of long-term oxidation of MeBr on NO₂-producing activity is more similar to what occurred when N. europaea lost approximately 90% of its NH₄⁺-dependent O₂ uptake activity during short-term cooxidation of trichloroethylene. In that case, very little NO₂-producing activity was observed after 8 h of incubation, presumably because the cells had suffered too much nonspecific damage during trichloroethylene oxidation (8).

Because cells exposed to MeBr exhibit about one-tenth the rate of NO₂ production that unexposed cells exhibit, it seems reasonable to conclude that approximately 10% of the cells survived the 24-h MeBr oxidation period and that the exponential recovery of NO₂ production probably reflected proliferation of the surviving cells. It is not clear, however, why development of NO₂ production by the cells exposed to MeBr lagged behind development of NO₂ production by the diluted, unexposed control cells when the two inocula were adjusted so that the initial activities were similar. It is possible that some of the residual NO₂ production by the cells exposed to MeBr originated from cells that were no longer capable of cell division. A recent study has shown that when methane-grown Methylocystis trichosporium OB3b oxidizes some chlorinated ethylenes, cell viability decreases more rapidly than the activity of methane monooxygenase decreases (26). Other studies performed in our laboratory have shown that AMO activity can be either upregulated or downregulated in response to NH₄⁺ availability (21, 22), that de novo protein synthesis is extremely limited in cells exposed to 10 mM NH₄⁺ at pH 7 (5), and that production of the mRNA transcript for AMO is limited when rates of NO₂ production are supported by 2 mM NH₄⁺ at pH 7.5 (19). The faster development of NO₂ production by the unexposed cells might have been due to upregulation of NH₃-oxidizing activity (21), and the cells exposed to MeBr might have lacked this ability.

Finally, during the initial optimum phase of cooxidation of 0.44 mM MeBr, the rate of NO₂ production declined to approximately 30% of the initial rate before any effect on MeBr oxidation was observed (Fig. 1), and the M/N ratio increased from 0.13 to 0.47. Although it is not known how MeBr oxidation could cause NH₃ oxidation to decrease while it allows MeBr oxidation to continue unabated, cell viability might decrease if reductant generation became insufficient to meet the combined needs of NH₃ oxidation, MeBr cooxidation, and the essential maintenance requirements of the cell.

By carrying out cooxidation experiments with moderately low cell densities before we conducted ecologically based studies, we identified a number of additional physiological and molecular biological questions worth pursuing with N. europaea. Additional studies will be required (i) to determine the mechanism responsible for the MeBr-induced decreases in NH₃-oxidizing activity and cell viability in N. europaea; (ii) to examine in more detail the sequence of events that occur during recovery of cells that have reached their cooxidative transformation capacity; and (iii) to determine if cooxidative activity interferes with regulation of AMO activity and gene regulation in response to NH₄⁺ availability.